Poxviral vaccines

ABSTRACT

The present application relates to novel administration regimens for poxviral vectors comprising nucleic acid constructs encoding antigenic proteins and invariant chains. In particular the use of said poxviral vectors for priming or for boosting an immune response is disclosed.

The present application relates to improved poxviral vaccines comprisingnucleic acid constructs encoding antigenic proteins and invariant chainsand novel administration regimens for such poxviral vectors. Inparticular the use of said poxviral vectors for priming or for boostingan immune response is disclosed.

BACKGROUND OF THE INVENTION

Infectious diseases are still a major threat to mankind. One way ofpreventing or treating infectious diseases is the artificial inductionof an immune response by vaccination which is the administration ofantigenic material to an individual such that an adaptive immuneresponse against the respective antigen is developed. The antigenicmaterial may be pathogens (e.g. microorganisms or viruses) which arestructurally intact but inactivated (i.e. non-infective) or which areattenuated (i.e. with reduced infectivity), or purified components ofthe pathogen that have been found to be highly immunogenic. Anotherapproach for inducing an immune response against a pathogen is theprovision of expression systems comprising one or more vector encodingimmunogenic proteins or peptides of the pathogen. Such vector may be inthe form of naked plasmid DNA, or the immunogenic proteins or peptidesmay be delivered by using viral vectors, for example on the basis ofmodified vaccinia viruses (e.g. Modified Vaccinia Ankara; MVA) oradenoviral vectors. Such expression systems have the advantage ofcomprising well-characterized components having a low sensitivityagainst environmental conditions.

It is a particular aim when developing vector based expression systemsthat the application of these expression systems to a patient elicits animmune response which is protective against the infection by therespective pathogen. However, although inducing an immunogenic responseagainst the pathogen, some expression systems are not able to elicit animmune response which is strong enough to fully protect againstinfections by the pathogen. Accordingly, there is still a need forimproved expression systems which are capable of inducing a protectiveimmune response against a pathogen as well as for novel administrationregimens of known expression systems which elicit enhanced immuneresponses.

Antigens are peptide fragments presented on the surface of antigenpresenting cells by MHC molecules. Antigens may be of foreign, i.e.pathogenic, origin or stem from the organism itself, the latter arereferred to as self- or auto antigens. There are two classes of MHCmolecules, MHC class I (MHC-I) and MHC-class-II (MHC-II). MHC-Imolecules present fragments of peptides which are synthesized within therespective cell. MHC-II molecules present fragments of peptides whichwere taken up by phagocytosis and subsequently digested in the endosome.Typically, MHC-II molecules are only expressed by “professional” antigenpresenting cells such as macrophages or dendritic cells. Antigens boundto MHC-II molecules are recognized by T-helper cells. The binding of theT-cell receptor of a T-helper cell to an antigen presented by a MHC-IImolecule, together with cytokines secreted by the antigen-presentingcells, induces the maturation of an immature T-helper cell of the TH₀phenotype into various types of effector cells.

The MHC-II molecules are membrane-bound receptors which are synthesizedin the endoplasmatic reticulum and leave the endoplasmatic reticulum ina MHC class II compartment. In order to prevent endogenous peptides,i.e. self-antigens, from binding to the MHC-II molecule, the nascentMHC-II molecule combines with another protein, the invariant chain,which blocks the peptide-binding cleft of the MHC-II molecule. When theMHC class II compartment fuses to a late endosome containingphagocytosed and degraded proteins, the invariant chain is cleaved toleave only the CLIP region bound to the MHC-II molecule. In a secondstep, CLIP is removed by an HLA-DM molecule leaving the MHC-II moleculefree to bind fragments of the foreign antigen. Said fragments arepresented on the surface of the antigen-presenting cell once the MHCclass II compartment fuses with the plasma membrane, thus presenting theforeign antigens to other cells, primarily T-helper cells.

It has been found previously (WO 2007/062656, which published as US2011/0293704 and is incorporated by reference for the purpose ofdisclosing invariant chain sequences) that the fusion of the invariantchain to an antigen which is comprised by an expression system used forvaccination increases the immune response against said antigen, if it isadministered with an adenovirus. Moreover, said adenoviral constructproved useful for priming an immune response in the context of aprime-boosting vaccination regimen (WO 2010/057501, which published asUS 2010/0278904 and is incorporated by reference for the purpose ofdisclosing invariant chain sequences and adenoviral vectors encodinginvariant chain sequences). The present inventors have surprisinglyfound that the immune response against a given antigen can be evenenhanced, if instead of an adenovirus a poxvirus is used for delivery ofthe invariant chain antigen fusion. In this way an immune response canbe generated. It is particularly surprising that the poxviral vectorselicited this effect also when used for priming an immune response.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a poxviral vectorcomprising a nucleic acid construct for use in priming or boosting animmune response, the nucleic acid construct comprising:

-   -   (i) a nucleic acid sequence encoding at least one invariant        chain operatively linked to    -   (ii) a nucleic acid encoding at least one antigenic protein or        antigenic fragment thereof    -   wherein the encoded invariant chain is operatively linked to the        N-terminus of the encoded antigenic protein or antigenic        fragment thereof.

In another aspect, the present invention relates to a vaccinecombination comprising:

-   -   (a) a poxviral vector comprising a nucleic acid construct the        nucleic acid construct comprising:        -   (i) a nucleic acid sequence encoding at least one invariant            chain operatively linked to        -   (ii) a nucleic acid encoding at least one antigenic protein            or antigenic fragment thereof    -   wherein the encoded invariant chain is operatively linked to the        N-terminus of the encoded antigenic protein or antigenic        fragment thereof; and    -   (b) a viral vector comprising a nucleic acid sequence encoding        at least a second antigenic protein or antigenic fragment        thereof or a second antigenic protein or antigenic fragment        thereof    -   wherein at least one epitope of the first antigenic protein or        antigenic fragment thereof is immunologically identical to the        second antigenic protein or fragment thereof.

In yet another aspect, the present invention relates to theabove-described vaccine combination, for use in a prime-boostvaccination regimen. Methods using such poxviral vectors andcombinations are also provided.

In a further aspect, the present invention relates to a poxviral vectorfor increasing the antigen-specific CD8+ T cell response in a subjectcomprising a nucleic acid construct for use in priming or boosting animmune response, the nucleic acid construct comprising:

-   -   (i) a nucleic acid sequence encoding at least one invariant        chain operatively linked to    -   (ii) a nucleic acid encoding at least one antigenic protein or        antigenic fragment thereof        -   wherein the encoded invariant chain is operatively linked to            the N-terminus of the encoded antigenic protein or antigenic            fragment thereof, and    -   wherein after administration to a subject the CD8+ T cell        response of the subject against the antigenic protein or        antigenic fragment thereof is increased as compared to the same        regimen without invariant chain.

In a further aspect, the invariant chain is p33 or a variant or fragmentthereof. In another aspect, the invariant chain is p35 or a variant orfragment thereof. In another aspect, the invariant chain is p41 or avariant or fragment thereof. In another aspect, the invariant chain isp43 or a variant or fragment thereof

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art.

For example, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Klbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. All definitions provided herein in thecontext of one aspect of the invention also apply to the other aspectsof the invention.

The problem underlying the present invention is solved by theembodiments characterized in the claims and in the description below.

In a first aspect the present invention relates to a poxviral vectorcomprising a nucleic acid construct for use in priming or boosting animmune response, the nucleic acid construct comprising:

-   -   (i) a nucleic acid sequence encoding at least one antigenic        protein or antigenic fragment thereof operatively linked to    -   (ii) a nucleic acid encoding at least one invariant chain.

As used herein, the term “poxviral vector” refers to a naturallyoccurring member of the family poxviridae or a viral vector derivedtherefrom which is capable of introducing the nucleic acid constructinto a cell of an individual. In the context of the present invention itis contemplated that the antigen and the invariant chain encoded by theintroduced nucleic acid construct are expressed within said cell uponintroduction by the poxviral vector.

The family poxviridae is characteristed by a genome consisting ofdouble-stranded DNA. Suitably, the poxviral vector belongs to thesubfamily chordopoxvirinae, more preferably to a genus in said subfamilyselected from the group consisting of orthopox, parapox, yatapox, avipox(preferably canarypox (ALVAC) or fowlpox (FPV)) and molluscipox. Evenmore preferably, the poxviral vector belongs to the orthopox and isselected from the group consisting of vaccinia virus, NYVAC (derivedfrom the Copenhagen strain of vaccinia), modified vaccinia Ankara (MVA),cowpoxvirus and monkeypox virus. Most preferably, the poxviral vector isMVA.

A description of MVA can be found in Mayr A, Stickl H, Müller HK, DannerK, Singer H. “The smallpox vaccination strain MVA: marker, geneticstructure, experience gained with the parenteral vaccination andbehavior in organisms with a debilitated defense mechanism. “Abstammung,Eigenschaften and Verwendung des attenuierten Vaccinia-Stammes MVA.”Zentralbl Bakteriol B. 1978 December; 167(5-6):375-90 and in Mayr, A.,Hochstein-Mintzel, V. & Stickl, H. (1975). Infection 3, 6-14.

MVA is a highly attenuated strain of vaccinia virus that underwentmultiple, fully characterised deletions during more than 570 passages inchick embryo fibroblast cells. These included host range genes and genesencoding cytokine receptors. The virus is unable to replicateefficiently in human and most other mammalian cells but the replicationdefect occurs at a late stage of virion assembly such that viral andrecombinant gene expression is unimpaired making MVA an efficient singleround expression vector incapable of causing infection in mammals.

In one embodiment, MVA is derived from the virus seed batch 460 MGobtained from 571th passage of Vaccinia Virus on CEF cells. In anotherembodiment, MVA is derived from the virus seed batch MVA 476 MG/14/78.In a further embodiment, MVA is derived or produced prior to 31 Dec.1978 and is free of prion contamination.

Further poxviral vectors for the use of the invention have propertiessimilar to MVA. In particular they are infectious but replicationincompetent in humans. Due to this trait, it may be necessary to expressproteins in trans for replication. Typically those proteins are stablyor transiently expressed in a viral producer cell line, thereby allowingreplication of the virus.

The term “nucleic acid” refers to a polymeric macromolecule made fromnucleotide monomers. Nucleotide monomers are composed of a nucleobase, afive-carbon sugar (such as but not limited to ribose or 2′-deoxyribose),and one to three phosphate groups. Typically, a polynucleotide is formedthrough phosphodiester bonds between the individual nucleotide monomers.In the context of the present invention nucleic acid molecules includebut are not limited to ribonucleic acid (RNA) and deoxyribonucleic acid(DNA). Moreover, the term “polynucleotide” also includes artificialanalogs of DNA or RNA, such as peptide nucleic acid (PNA).

The term “nucleic acid construct” refers to a nucleic acid which encodesat least one antigenic protein and at least one invariant chain.Suitably, said nucleic acid additionally comprises elements which directtranscription and translation of the polypeptides encoded by the nucleicacid construct. Such elements include promoter and enhancer elements todirect transcription of mRNA in a cell-free or a cell-based basedsystem, for example a cell-based system. Suitably such promoter and/orenhancer is an endogenous promoter and/or enhancer of the poxviralvector. If the acid construct is provided as translatable RNA, it isenvisioned that the nucleic acid construct comprises those elements thatare necessary for translation and/or stabilization of RNAs encoding theat least one immunogenic polypeptide, e.g. polyA-tail, IRES, capstructures etc.

The term “substantially similar” if used in relation to nucleic acidsequences or amino acid sequences refers to a degree of sequenceidentity of more than 80%, more than 85%, more than 90%, more than 95%,more than 98%, or more than 99% of the respectively indicated referencenucleotide or amino acid sequence.

Residues in two or more polypeptides are said to “correspond” to eachother if the residues or group of residues occupy (an) analogousposition(s) in the polypeptide structures. As is well known in the art,analogous positions in two or more polypeptides can be determined byaligning the polypeptide sequences based on amino acid sequence orstructural similarities. Such alignment tools are well known to theperson skilled in the art and can be, for example, obtained on the WorldWide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align(www.ebi.ac.uk/emboss/align/index.html) using standard settings, forexample for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, GapExtend 0.5. Those skilled in the art understand that it may be necessaryto introduce gaps in either sequence to produce a satisfactoryalignment. Residues are said to “correspond” if the residues are alignedin the best sequence alignment. The “best sequence alignment” betweentwo polypeptides is defined as the alignment that produces the largestnumber of aligned identical residues. The “region of best sequencealignment” ends and, thus, determines the metes and bounds of the lengthof the comparison sequence for the purpose of the determination of thesimilarity score, if the sequence similarity or identity between twoaligned sequences drops to less than 30%, less than 20%, or less than10% over a length of 10, 20 or 30 amino acids.

As outlined above it is contemplated that the vector of the presentinvention is a poxviral vector. Thus, if said poxviral vector isreplication competent, the nucleic acid construct is comprised by alarger nucleic acid molecule which additionally includes nucleic acidsequences which are required for the replication of the viral vectorand/or regulatory elements directing expression of the polypeptideencoded by the nucleic acid construct.

In one embodiment of the present invention the antigenic protein orantigenic fragment thereof and the invariant chain are comprised by asingle open reading frame so that transcription and translation of saidopen reading frame (“ORF”) results in a fusion protein comprising theantigenic protein or antigenic fragment thereof and the invariant chain.When the nucleic acids encoding the antigenic protein or antigenicfragment thereof and the invariant chain are comprised by a single ORF,they are said to be “operatively linked”. Such operatively-linkednucleic acids may be directly linked, i.e., the 3′ end of the firstpolynucleotide is directly adjacent to the 5′ end of the second sequencewith no intervening nucleic acids. Alternatively, the ORFs may beindirectly linked such that there are intervening nucleic acids. Forexample, the intervening nucleic acids may be noncoding or may encode anamino acid sequence, for example a peptide linker. Operatively-linkednucleic acids may encode polypeptides that are directly linked, i.e.,the carboxy-terminus (“C-terminus”) of one encoded polypeptide isdirectly adjacent to the amino-terminus (“N-terminus”) of a secondencoded polypeptide. Alternatively, operatively-linked nucleic acids mayencode indirectly linked polypeptides such that there are interveningamino acids between the encoded polypeptides, for example a linker.

The term “open reading frame” or ORF refers to a sequence ofnucleotides, that can be translated into amino acids. Typically, such anORF contains a start codon, a subsequent region usually having a lengthwhich is a multiple of 3 nucleotides, but does not contain a stop codon(TAG, TAA, TGA, UAG, UAA, or UGA) in the given reading frame. Typically,ORFs occur naturally or are constructed artificially, i.e. bygene-technological means. An ORF codes for a protein where the aminoacids into which it can be translated form a peptide-linked chain.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein and refer to any peptide-linked chain of aminoacids, regardless of length, co-translational or post-translationalmodification.

The term “post-translational” used herein refers to events that occurafter the translation of a nucleotide triplet into an amino acid and theformation of a peptide bond to the preceding amino acid in the sequence.Such post-translational events may occur after the entire polypeptidewas formed or already during the translation process on those parts ofthe polypeptide that have already been translated. Post-translationalevents typically alter or modify the chemical or structural propertiesof the resultant polypeptide. Examples of post-translational eventsinclude but are not limited to events such as glycosylation orphosphorylation of amino acids, or cleavage of the peptide chain, e.g.by an endopeptidase.

The term “co-translational” used herein refers to events that occurduring the translation process of a nucleotide triplet into an aminoacid chain. Those events typically alter or modify the chemical orstructural properties of the resultant amino acid chain. Examples ofco-translational events include but are not limited to events that maystop the translation process entirely or interrupt the peptide bondformation resulting in two discreet translation products.

Proteins usable in the present invention (including protein derivatives,protein variants, protein fragments, protein segments, protein epitopesand protein domains) can be further modified by chemical modification.Hence, such a chemically modified polypeptide may comprise chemicalgroups other than the residues found in the 20 naturally occurring aminoacids. Examples of such other chemical groups include without limitationglycosylated amino acids and phosphorylated amino acids. Chemicalmodifications of a polypeptide may provide advantageous properties ascompared to the parent polypeptide, e.g. one or more of enhancedstability, increased biological half-life, or increased watersolubility. Chemical modifications applicable to the variants usable inthe present invention include without limitation: PEGylation,glycosylation of non-glycosylated parent polypeptides, or themodification of the glycosylation pattern present in the parentpolypeptide. Such chemical modifications applicable to the variantsusable in the present invention may occur co- or post-translational.

An “antigenic protein” as referred to in the present application is apolypeptide as defined above which contains at least one epitope. An“antigenic fragment” of an antigenic protein is a partial sequence ofsaid antigenic protein comprising at least one epitope. For immunizationpurposes only those parts of a protein are relevant which elicit animmune response. Therefore, the nucleic acid construct does not need toencode the full-length antigenic protein as it is found in a pathogen ora cancer cell. A shortened fragment of such a protein is sufficient aslong as its amino acid sequence comprises the epitope or epitopesresponsible for the recognition of the antigenic protein by the immunesystem.

The term “epitope” also known as antigenic determinant, as used in thecontext of the present invention is that part of a polypeptide which isrecognized by the immune system. Suitably, this recognition is mediatedby the binding of antibodies, B cells, or T cells to the epitope inquestion. The epitopes bound by antibodies or B cells are referred to as“B cell epitopes” and the epitopes bound by T cells are referred to as“T cell epitopes”. In this context, the term “binding” relates to aspecific binding, which is defined as a binding with an associationconstant between the antibody or T cell receptor (TCR) and therespective epitope of 1×10⁵ M⁻¹ or higher, or of 1×10⁶ M⁻¹, 1×10⁷ M⁻¹,1×10⁸ M⁻¹ or higher. The skilled person is well aware how to determinethe association constant (see e.g. Caoili, S. E. (2012) Advances inBioinformatics Vol. 2012). Suitably, the specific binding of antibodiesto an epitope is mediated by the Fab (fragment, antigen binding) regionof the antibody, specific binding of a B-cell is mediated by the Fabregion of the antibody comprised by the B-cell receptor and specificbinding of a T-cell is mediated by the variable (V) region of the T-cellreceptor. T cell epitopes are presented on the surface of an antigenpresenting cell, where they are bound to Major Histocompatiblilty (MHC)molecules. There are at least three different classes of MHC moleculestermed MHC class I, II and III molecules, respectively. Epitopespresented through the MHC-I pathway elicit a response by cytotoxic Tlymphocytes (CD8⁺ cells), while epitopes presented through the MHC-IIpathway elicit a response by T-helper cells (CD4⁺ cells). T cellepitopes presented by MHC Class I molecules are typically peptidesbetween 8 and 11 amino acids in length and T cell epitopes presented byMHC Class II molecules are typically peptides between 13 and 17 aminoacids in length. MHC Class III molecules also present non-peptidicepitopes as glycolipids. Accordingly, the term “T cell epitope” refersto a 8 to 11 or 13 to 17 amino acid long peptide that can be presentedby either a MHC Class I or MHC Class II molecule.

Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. The term “epitope” refers toconformational as well as non-conformational epitopes. Conformationaland non-conformational epitopes are distinguished in that the binding tothe former but not the latter is lost in the presence of denaturingsolvents. T cell epitopes are non-conformational, i.e. they are linear,while B cell epitopes can be conformational or non-conformational.Linear B-cell epitopes typically vary between 5 to 20 amino acids inlength.

An antigenic protein according to the present invention is derived froma pathogen selected from the group consisting of viruses, bacteria,protozoa and multicellular parasites. In an alternative embodiment ofthe present invention the antigenic protein is a polypeptide or fragmentof a polypeptide expressed by a cancer cell.

In one embodiment, the antigenic protein is not derived from HCV. Forexample, one aspect provides a poxviral vector comprising a nucleic acidconstruct the nucleic acid construct comprising (i) a nucleic acidsequence encoding at least one invariant chain operatively linked to(ii) a nucleic acid encoding at least one antigenic protein or antigenicfragment thereof, wherein the C-terminus of the encoded invariant chainis directly or indirectly linked to the N-terminus of the encodedantigenic protein or antigenic fragment thereof, and wherein theantigenic protein or fragment thereof is not derived from HCV. In afurther embodiment, the antigenic protein or fragment thereof is notHCV-NS.

In another embodiment, the antigenic protein is not derived frommalaria. For example, one aspect provides a poxviral vector comprising anucleic acid construct the nucleic acid construct comprising (i) anucleic acid sequence encoding at least one invariant chain operativelylinked to (ii) a nucleic acid encoding at least one antigenic protein orantigenic fragment thereof, wherein the C-terminus of the encodedinvariant chain is directly or indirectly linked to the N-terminus ofthe encoded antigenic protein or antigenic fragment thereof, and whereinthe antigenic protein or fragment thereof is not derived from malaria.In a further embodiment, the antigenic protein or fragment thereof isnot ME-TRAP.

Antigenic proteins or antigenic fragments thereof induce a B-cellresponse or a T-cell response or a B-cell response and a T-cellresponse. Accordingly, antigenic proteins or antigenic fragmentscomprise at least one T cell epitope and/or at least one B cell epitope.

In a certain exemplary embodiment of the present invention, theantigenic protein encoded by the vector is derived from hepatitis Cvirus (HCV). The HCV genome consists of a single RNA strand about 9.5 kbin length which encodes a precursor polyprotein of about 3000 aminoacids. (Choo et al. (1989) Science 244, 362-364; Choo et al. (1989)Science 244, 359-362; Takamizawa et al. (1991) J. Virol. 65, 1105-1113)The HCV polyprotein contains the viral proteins in the order:C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Individual viral proteins areproduced by proteolysis of the HCV polyprotein. Host cell proteasesrelease the putative structural proteins C, E1, E2, and p7, and createthe N-terminus of NS2 at amino acid 810. (Mizushima et al. (1994) J.Virol. 65, 2731-2734; Hijikata et al. (1993) P.N.A.S. USA 90,10773-10777)

The non-structural proteins NS3, NS4A, NS4B, NS5A and NS5B presumablyform the virus replication machinery and are released from thepolyprotein. A zinc-dependent protease associated with NS2 and theN-terminus of NS3 is responsible for cleavage between NS2 and NS3.(Grakoui et al. (1993) J. Virol. 67, 1385-1395, Hijikata et al. (1993)P.N.A.S. USA 90, 10773-10777,) A distinct serine protease located in theN-terminal domain of NS3 is responsible for proteolytic cleavages at theNS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A7NS5B junctions. (Bartenschlageret al. (1993) J. Virol. 67, 3835-3844; Grakoui et al., (1993) Proc.Natl. Acad. Sci. USA 90, 10583-10587, Tomei et al. (1993) Virol. 67,4017-4026) NS4A provides a cofactor for NS3 activity. (Failla et al.(1994) J. Virol. 68, 3753-3760; De Francesco et al, U.S. Pat. No.5,739,002.). NS5A is a highly phosphorylated protein conferringinterferon resistance. (De Francesco et al., (2000) Semin Liver Dis.,20(1), 69-83; Pawlotsky (1999) Viral Hepat. Suppl. 1, 47-48). NS5Bprovides an RNA-dependent RNA polymerase. (De Francesco et al,International Publication Number WO 96/37619, Behrens et al, EMBO 15,12-22, 1996, Lohmann et al., Virology 249, 108-118, 1998.)

In one non-limiting exemplary embodiment, the antigenic protein is aMet-NS3-NS4A-NS4B-NS5A-NS5B polypeptide containing an inactive NS5BRNA-dependent RNA polymerase region. For example, said antigenic proteinhas an amino acid sequence substantially similar to the sequence definedby SEQ ID NO: 11 and has sufficient protease activity to process itselfto produce at least a polypeptide substantially similar to the NS5Bregion present in SEQ ID NO: 11. The sequence of this antigenic proteinhas been published in WO 2003/031588, which published as US 2004/0247615and is incorporated by reference for the purpose of disclosing HCVpolypeptides. The produced polypeptide corresponding to NS5B isenzymatically inactive. In a further embodiment, the HCV polypeptide hassufficient protease activity to produce polypeptides substantiallysimilar to the NS3, NS4A, NS4B, NS5A, and NS5B regions present in SEQ IDNO: 11.

In one embodiment, the degree of sequence identity to the sequenceaccording to SEQ ID NO: 11 is more than 80%, more than 85%, more than90%, more than 95% or more than 98% of the sequence defined by SEQ IDNO: 11. However, in some embodiments, the sequence of the antigenicprotein has more than 99% sequence identity to the sequence defined bySEQ ID NO: 11 or is identical to it.

In one embodiment, the invariant chain is operatively linked at theN-terminus of the at least one antigenic protein. In a further aspect,the invariant chain is operatively linked to an HCV antigen. In afurther aspect, SEQ ID NO:1 is operatively linked to SEQ ID NO:11. In afurther aspect, SEQ ID NO:1 is operatively linked at the N-terminus ofSEQ ID NO:11. In a further embodiment, the invariant chain is encoded bya nucleic acid operatively linked to a nucleic acid of at least oneantigenic protein or fragment thereof.

The term “invariant chain”, also known as “li” or “CD74” refers to anon-polymorphic type II integral membrane protein. The protein hasmultiple functions in lymphocyte maturation and adaptive immuneresponses; in particular li ensures the targeting of newly synthesizedMCH II to the endocytic pathway, where the complex can meet antigenicpeptides. (Pieters J. (1997) Curr. Opin. Immunol., 9: 8996).Additionally, li has been shown to function as an MHC class I chaperone(Morris et al. (2004) Immunol. Res., 30: 171-179) and, by its endosomaltargeting sequence, to facilitate stimulation of CD4⁺, but not CD8⁺T-cells directed against covalently linked antigen (Diebold et al.(2001) Gene Ther. 8: 487-493).

For human invariant chain four different isoforms are known, generallytermed p33, p35, p41 and p43 (Strubin et al., 1986, EMBO Journal, 5:3483-3488). SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the amino acidsequence and the nucleic acid sequence of human invariant chain p35isoform. With respect to human p33 and p41 the human p35 and p43isoforms contain an additional 16 residues at the N-terminus due toalternative initiation of translation. Compared to human p33 and p35 thehuman p41 and p43 isoforms comprise an additional domain (alternativesplicing of exon 6b) inserted in frame in the C-terminal region of theinvariant chain. SEQ ID NO: 5 and SEQ ID NO: 6 correspond to the aminoacid sequence and the nucleic acid sequence of human invariant chain p43isoform. The sequence of an additional human isoform c lacking two exonsrelative to human p33 and p35 is available in Genbank (AccessionBC024272). SEQ ID NO: 9 and SEQ ID NO: 10 correspond to the amino acidsequence and the nucleic acid sequence of human invariant chain cisoform.

TABLE 1 Overview over the variants of human invariant chain 16 AA atAdditional SEQ ID NO: Isoform N-terminus domain (peptide, nucleic acid)P33 − − — P35 + − 1, 2 P41 − + — P43 + + 5, 6 c + −  9, 10

For murine invariant chain only two isoforms (p31 and p41) are knowncorresponding to the human invariant chain isoforms p33 and p41. SEQ IDNO: 3 and SEQ ID NO: 4 correspond to the amino acid sequence and thenucleic acid sequence of murine invariant chain p31 isoform. SEQ ID NO:7 and SEQ ID NO: 8 correspond to the amino acid sequence and the nucleicacid sequence of murine invariant chain p41 isoform. A schematicoverview over the different isoforms is shown in FIG. 4.

In one embodiment, the invariant chain used in the present invention issubstantially similar to the invariant chain according to SEQ ID NO: 1or 3.

The invariant chain comprises several domains: a cytosolic domain whichincludes a sorting (targeting) peptide (also known as the lysosomaltargeting sequence) (positions 17 to 46 in human invariant chain SEQ IDNO: 1, positions 1 to 29 in the murine invariant chain SEQ ID NO: 3)preceded by an ER retention signal in the human invariant chain p35 andp43 variants (positions 1 to 16 in human invariant chain SEQ ID NO: 1),a transmembrane domain (signal anchor, positions 47 to 72 in humaninvariant chain SEQ ID NO: 1, positions 30 to 55 in the murine invariantchain SEQ ID NO: 3), and a luminal domain which in itself comprises aKEY region (positions 93 to 96 in human invariant chain SEQ ID NO: 1,positions 76 to 79 in the murine invariant chain SEQ ID NO: 3), anadjacent CLIP region (positions 97 to 120 in human invariant chain SEQID NO 1, positions 80 to 103 in the murine invariant chain SEQ ID NO:3). The CLIP region comprises a core CLIP peptide (positions 103 to 117in human invariant chain SEQ ID NO: 1, positions 86 to 100 in the murineinvariant chain SEQ ID NO: 3) and a trimerization domain (positions 134to 208 in human invariant chain SEQ ID NO: 1, positions 117 to 191 inthe murine invariant chain SEQ ID NO: 3; Mittendorf et al., (2009)Expert Opin. Biol. Ther., 9:71-78; Strumptner-Cuvelette and Benaroch,2002, Biochem. Biophys. Acta, 1542: 1-13). The remainder of the luminaldomain comprises two highly flexible regions situated between thetransmembrane and KEY region (positions 73 to 92 in human invariantchain SEQ ID NO: 1, positions 56 to 75 in the murine invariant chain SEQID NO: 3) or downstream the trimerization domain (positions 209 to 232in human invariant chain SEQ ID NO: 1, positions 192 to 215 in themurine invariant chain SEQ ID NO: 3). Invariant chain has beencharacterized in several organisms such as chicken, cow, dog, mouse, ratand human.

In one embodiment, the invariant chain is of vertebrate origin, of avianor mammalian origin, or further, it is selected from the groupconsisting of invariant chains derived from chicken, cow, dog, mouse,rat, non-human primate and human. In a further embodiment, it is ofhuman or murine origin, for example, the human invariant chain has anamino acid sequence as defined by SEQ ID NO: 1. Said polypeptide is inone embodiment encoded by a nucleic acid sequence as given in SEQ ID NO:2. In another embodiment, the murine invariant chain has an amino acidsequence as defined by SEQ ID NO: 3. Said polypeptide is in oneembodiment encoded by a nucleic acid sequence as given in SEQ ID NO: 4.

The term “invariant chain” also comprises variants of theabove-described polypeptides characterized by deletions of parts of theamino acid sequences of naturally occurring invariant chains or ofinvariant chains substantially similar to the naturally occurringinvariant chains or by their substitution with other sequences.Exemplary variants are given below.

In one particular variant of the invariant chain, the endogenousKEY-region which consists of the LRMK amino acid residues is deleted oris substituted by a different amino acid sequence. For example, the LRMKamino acid residues or corresponding residues are deleted. Deletion ofthe LRMK amino acid residues may be complete (involving all LRMK aminoacid residues) or partial (involving at least one LRMK amino acidresidue). Complete deletion of all LRMK amino acid residues isenvisioned. Further, at least one or all of the LRMK amino acid residuesare substituted by different amino acid residues.

In yet another exemplary variant the methionines in positions 107 and115 of the human invariant chain according to SEQ ID NO: 1 or themethionines in positions 90 and 98 of the murine invariant chainaccording to SEQ ID NO: 3 or the methionines corresponding to thesepositions in other invariant chains are substituted by other aminoacids. Suitably, the methionine is substituted.

In yet another exemplary variant, the invariant chain is N-terminallytruncated, for example to such an extent that the N-terminus up to thetransmembrane region is removed. Accordingly, in a further embodiment,the invariant chain according to Seq ID NO: 1 46 amino acids or less ofthe N-terminus are truncated, 41 amino acids or less are truncated, or36 amino acids or less are truncated. Accordingly it is alsocontemplated that for the invariant chain according to Seq ID NO: 3 30amino acids or less of the N-terminus are truncated, 25 amino acids orless are truncated, or 20 amino acids or less are truncated. For oneembodiment of the invariant chain according to SEQ ID NO: 1, the first16 amino acid residues of the human invariant chain are deleted. It isalso possible that at least one, but not all of the first 16 amino acidresidues are deleted. Furthermore, it is possible that at least one, orall of the first 16 amino acid residues of the human invariant chain(SEQ ID NO: 1) are substituted by other amino acid residues.

In yet another variant, at least one signal peptide for expression inthe lumen of the endoplasmatic reticulum is added to the N-terminus ofthe invariant chain, for example to an N-terminally truncated version ofthe invariant chain which—due to the N-terminal truncation—lacks thetransmembrane region.

In yet another variant of the invariant chain at least one CLIP regionis added to or replaces the endogenous CLIP region of the respectiveinvariant chain. In the human invariant chain according to SEQ ID NO: 1the CLIP region spans positions 97 to 120 and in the murine invariantchain according to SEQ ID NO 3 it spans positions 80 to 103. Thus, theskilled person can easily determine the amino acid residuescorresponding to the CLIP region in the invariant chain according to SEQID NO: 1 and 3. In a further embodiment, the complete endogenous CLIPregion is deleted or replaced. However, the deletion or replacement ofat least one amino acid residue belonging to the endogenous CLIP regionis also contemplated.

The term “invariant chain” also refers to fragments of the invariantchains and their variants described above, for example, the invariantchains having amino acid sequences according to SEQ ID NO: 1 or 3 orencoded by nucleic acid sequences according SEQ ID NO: 2 or 4. It is tobe understood that due to the degenerated nature of the genetic code,one amino acid may be encoded by more than one codon. For example, theamino acid isoleucine may be encoded by the codons AUU, AUC or AUA.Therefore, the present invention also encompasses all variants of theaforementioned nucleic acid sequences which encode the amino acidsequences defined by SEQ ID NOs: 1 or 3 irrespective of the specificnucleic acid sequence. Since different organisms utilize differentcodons with different efficiency, it may be advantageous to adapt thecodon usage in a nucleotide sequence to the intended host organism.

In one embodiment, the fragment is a fragment of at least 40, 50, 60,70, 80, 90, 10, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210amino acid residues of a wild-type invariant chain or a variant thereofas defined above.

Moreover, the term “invariant chain” refers to polypeptides having atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8% or 99.9% sequence identity with any of the above describedwild-type invariant chains, variants or fragments thereof. Methods fordetermining the sequence identity between two different polypeptides arewell known in the art. The similarity of nucleotide and amino acidsequences, i.e. the percentage of sequence identity, can be determinedvia sequence alignments. Such alignments can be carried out with severalart-known algorithms, for example with the mathematical algorithm ofKarlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA90: 5873-5877), with hmmalign (HMMER package, hmmer.wustl.edu/) or withthe CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994) Nucleic Acids Res. 22, 4673-80) available e.g. onwww.ebi.ac.uk/Tools/clusta1w/ or onwww.ebi.ac.uk/Tools/clustalw2/index.html or onnpsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clusta1w.html.Preferred parameters used are the default parameters as they are set onwww.ebi.ac.uk/Tools/clusta1w/ orwww.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity(sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ(or BlastX). A similar algorithm is incorporated into the BLASTN andBLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410.

To obtain gapped alignments for comparative purposes, Gapped BLAST isutilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs are used. Sequence matchinganalysis may be supplemented by established homology mapping techniqueslike Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identityare referred to in the present application, these percentages arecalculated in relation to the full length of the longer sequence, if notspecifically indicated otherwise.

In one embodiment, the invariant chain is p33 or variants or fragmentsthereof. In a further embodiment, the invariant chain is a variant ofp33. In yet another further embodiment, the invariant chain is afragment of p33. In another embodiment, the invariant chain is p35 orvariants or fragments thereof. In a further embodiment, the invariantchain is a variant of p35. In yet another further embodiment, theinvariant chain is a fragment of p35. In another embodiment, theinvariant chain is p41 or variants or fragments thereof. In a furtherembodiment, the invariant chain is a variant of p41. In yet anotherfurther embodiment, the invariant chain is a fragment of p41. In anotherembodiment, the invariant chain is p43 or variants or fragments thereof.In a further embodiment, the invariant chain is a variant of p43. In yetanother further embodiment, the invariant chain is a fragment of p43.

The term “priming an immune response” refers to the first encounter ofthe immune system with the at least one antigenic protein or antigenicfragment thereof and the subsequent induction of an antigen-specificimmune response within a defined period of time. Said period of time is,for example, at least 1 year, at least 2 years, at least 3 years, atleast 5 years or at least 10 years prior to the priming. In oneembodiment, encounters of the individual's or subject's immune systemwith the antigenic protein or antigenic fragment thereof of which do notinduce an antigen-specific immune response are not considered as“priming an immune response”. For example, encounters of theindividual's immune system with the antigenic protein or antigenicfragment thereof which do not induce lasting immunity are not consideredas “priming an immune response” according to the present invention. In afurther embodiment, the induction of lasting immunity is mediated by thegeneration of memory B cells and/or memory T cells. In the case ofcancer, for example, a specific antigen may be expressed by the cancercells without eliciting an immune response. The mere presence of thisantigen is not a “priming of an immune response” against said antigen asunderstood by the present application. In one embodiment, the individualor subject has not been deliberately immunized with the antigenicprotein or antigenic fragment thereof or a vector comprising a nucleicacid encoding such a protein or fragment with the aim of treating orpreventing a disease in the period of time given before.

However, the term “priming an immune response” refers to any previouscontact of the individual with a pathogen naturally comprising saidantigenic protein or antigenic fragment thereof provided that saidcontact was not artificially induced for medical purposes. In particularit is envisaged by the present application that the individual mayalready have been infected with the aforementioned pathogen providedthat said infection was not artificially induced for medical purposes.At the time the priming of the immune response takes place, theinfection of the individual may still be present or it may already havebeen eliminated. Similarly, in the case of cancer, it is envisaged thatthe individual or subject to be immunized with the poxviral vector ofthe present invention already suffers from the cancer expressing theantigenic protein or antigenic fragment thereof which is comprised bythe nucleic acid construct of the present invention.

The patient or subject to be immunized with a poxviral vector accordingto the present invention is, for example, a mammal or a bird, morespecifically a primate, mouse, rat, sheep, goat, cow, pig, horse, goose,chicken, duck or turkey and, most specifically, a human.

The poxviral vector comprising a nucleic acid construct as defined aboveis, for example, used in a prime-boost vaccination regimen.

In many cases, a single administration of a vaccine is not sufficient togenerate the number of long-lasting immune cells which is required foreffective protection in case of future infection of the pathogen inquestion, protect against diseases including tumour diseases or fortherapeutically treating a disease, like tumour disease. Consequently,repeated challenge with a biological preparation specific for a specificpathogen or disease is required in order to establish lasting andprotective immunity against said pathogen or disease or to cure a givendisease. An administration regimen comprising the repeatedadministration of a vaccine directed against the same pathogen ordisease is referred to in the present application as “prime-boostvaccination regimen”. In one embodiment, a prime-boost vaccinationregimen involves at least two administrations of a vaccine or vaccinecomposition directed against a specific pathogen, group of pathogens ordiseases. The first administration of the vaccine is referred to as“priming” and any subsequent administration of the same vaccine or avaccine directed against the same pathogen as the first vaccine isreferred to as “boosting”. Thus, in a further embodiment of the presentinvention the prime-boosting vaccination regimen involves oneadministration of the vaccine for priming the immune response and atleast one subsequent administration for boosting the immune response. Itis to be understood that 2, 3, 4 or even 5 administrations for boostingthe immune response are also contemplated by the present invention.

The period of time between prime and boost is, optionally, 1 week, 2weeks, 4 weeks, 6 weeks or 8 weeks. More particularly, it is 4 weeks or8 weeks. If more than one boost is performed, the subsequent boost isadministered 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks after thepreceding boost. For example, the interval between any two boosts is 4weeks or 8 weeks.

Prime-boost vaccination regimens may be homologous or heterologous. Inhomologous prime-boost regimens both the priming and the at least oneboosting is performed using the same means of administration of theantigenic protein or antigenic fragment thereof, i.e. priming andboosting are performed using a polypeptide or priming and boosting areperformed using a nucleic acid construct comprised by the same vector.In the context of the present invention a homologous prime-boostvaccination regimen would comprise the use of the poxviral vector of theinvention both for priming as well as for boosting the immune response.A heterologous prime-boosting regimen involves the use of differentmeans for priming and for boosting the immune response. In the contextof the present invention, a heterologous prime-boosting regimen wouldcomprise a poxviral vector as described above for the priming of animmune response and a different vector or a peptide vaccine for theboosting of the immune response.

Alternatively, a heterologous prime-boosting regimen would comprise adifferent vector or a peptide vaccine for the priming of an immuneresponse and a poxviral vector as described above for the boosting ofthe immune response.

In one embodiment of the present invention the prime-boostingvaccination regimen is homologous.

In another embodiment of the present invention the prime-boostingvaccination regimen is heterologous.

In one heterologous prime boosting regimen a poxviral vector asdescribed above is used for the boosting of the immune response and adifferent vector or a peptide vaccine is used for the priming of theimmune response. In another embodiment, heterologous prime boostingregimen, a poxviral vector as described above is used for the priming ofthe immune response and a different vector or a peptide vaccine is usedfor the boosting of the immune response.

In another embodiment the heterologous prime-boosting regimen wouldcomprise an adenovirus vector for the priming of an immune response anda poxviral vector as described above for the boosting of the immuneresponse.

In yet another embodiment, the heterologous prime boosting regimen wouldcomprise an poxviral vector as described above for the priming of animmune response and a adenovirual vector for the boosting of the immuneresponse.

For all prime-boosting vaccination regimens it is envisioned that theantigenic proteins or antigenic peptides used for boosting the immuneresponse are immunologically identical to the antigenic protein orantigenic fragment thereof used for priming the immune response. It isto be understood that the antigenic protein or antigenic fragmentthereof may be administered as a polypeptide (“peptide vaccine”) or thatit may be encoded by a nucleic acid molecule administered to theindividual in question. In the latter case, the antigenic protein orantigenic polypeptide which elicits the desired immune response isexpressed in the cells of immunized individual.

Two or more antigenic proteins or antigenic fragments thereof are“immunologically identical” if they are recognized by the same antibody,T-cell or B-cell. The recognition of two or more immunogenicpolypeptides by the same antibody, T-cell or B-cell is also known as“cross reactivity” of said antibody, T-cell or B-cell. In oneembodiment, the recognition of two or more immunologically identicalpolypeptides by the same antibody, T-cell or B-cell is due to thepresence of identical or similar epitopes in all polypeptides. Similarepitopes share enough structural and/or charge characteristics to bebound by the Fab region of the same antibody or B-cell receptor or bythe V region of the same T-cell receptor. The binding characteristics ofan antibody, T-cell receptor or B-cell receptor are, for example,defined by the binding affinity of the receptor to the epitope inquestion. Two immunogenic polypeptides are “immunologically identical”as understood by the present application if the affinity constant ofpolypeptide with the lower affinity constant is at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95% or at least 98% of the affinity constant of thepolypeptide with the higher affinity constant. Methods for determiningthe binding affinity of a polypeptide to a receptor such as equilibriumdialysis or enzyme linked immunosorbent assay (ELISA) are well known inthe art.

In one embodiment, two or more “immunologically identical” polypeptidescomprise at least on identical epitope. The strongest vaccinationeffects can usually be obtained, if the immunogenic polypeptidescomprise identical epitopes or if they have an identical amino acidsequence.

In one embodiment, the use of the poxviral vector as described above forthe priming or boosting of an immune response will establish protectiveimmunity against a pathogen or disease or will lead to eradication ofthe disease.

In one embodiment, the use of the poxviral vector as described hereinfor the priming or boosting of an immune response will increase theantigen-specific CD8+ T cell response as compared to the same regimenwithout invariant chain.

In one embodiment, the poxviral vector is administered via intranasal,intramuscular, subcutaneous, intradermal, intragastric, oral and topicalroutes.

An “intranasal administration” is the administration of a vector of thepresent invention to the mucosa of the complete respiratory tractincluding the lung. More particularly, the composition is administeredto the mucosa of the nose. In one embodiment, an intranasaladministration is achieved by means of instillation, spray or aerosol.In a further embodiment, said administration does not involveperforation of the mucosa by mechanical means such as a needle.

The term “intramuscular administration” refers to the injection of avector into any muscle of an individual. Exemplary intramuscularinjections are administered into the deltoid, vastus lateralis or theventrogluteal and dorsogluteal areas.

The term “subcutaneous administration” refers to the injection of avector into the hypodermis.

The term “intradermal administration” refers to the injection of avector into the dermis between the layers of the skin.

The term “oral administration” refers to the administration of a vectorvia the mouth to the gastric system.

A “topical administration” is the administration of the vector to anypart of the skin without penetrating the skin with a needle or acomparable device. The vector may also be administered topically to themucosa of the mouth, nose, genital region and rectum.

In another aspect, the present invention relates to a vaccinecombination comprising:

-   -   (a) a poxviral vector comprising a nucleic acid construct, the        nucleic acid construct comprising:        -   (i) a nucleic acid sequence encoding at least one invariant            chain operatively linked to        -   (ii) a nucleic acid encoding at least one antigenic protein            or antigenic fragment thereof wherein the encoded invariant            chain is operatively linked to the N-terminus of the encoded            antigenic protein or antigenic fragment thereof, and    -   wherein after administration to a subject the CD8+ T cell        response of the subject against the antigenic protein or        antigenic fragment thereof is increased as compared to the same        regimen without invariant chain.    -   (b) a vector comprising a nucleic acid sequence encoding at        least a second antigenic protein or antigenic fragment thereof    -   or        -   a second antigenic protein or antigenic fragment thereof    -   or        -   viral like particles

wherein at least one epitope of the first antigenic protein or antigenicfragment thereof is immunologically identical to the at least secondantigenic protein or fragment thereof.

The term “vaccine” refers to a biological preparation which induces orimproves immunity to a specific disease. Said preparation may comprise akilled or an attenuated living pathogen. It may also comprise one ormore compounds derived from a pathogen suitable for eliciting an immuneresponse. In one embodiment, said compound is a polypeptide which issubstantially identical or immunologically identical to a polypeptide ofsaid pathogen. In another embodiment, the vaccine comprises a nucleicacid construct which encodes an immunogenic polypeptide which issubstantially identical or immunologically identical to a polypeptide ofsaid pathogen. In the latter case, it is also contemplated that thepolypeptide is expressed in the individual treated with the vaccine. Theprinciple underlying vaccination is the generation of an immunological“memory”. Challenging an individual's immune system with a vaccineinduces the formation and/or propagation of immune cells whichspecifically recognize the compound comprised by the vaccine. At least apart of said immune cells remains viable for a period of time which canextend to 10, 20 or 30 years after vaccination. If the individual'simmune system encounters the pathogen from which the compound capable ofeliciting an immune response was derived within the aforementionedperiod of time, the immune cells generated by vaccination arereactivated and enhance the immune response against the pathogen ascompared to the immune response of an individual which has not beenchallenged with the vaccine and encounters immunogenic compounds of thepathogen for the first time.

As used herein, the term “vector” refers to at least one polynucleotideor to a mixture of at least one polynucleotide and at least one proteinwhich is capable of introducing the polynucleotide comprised thereininto a cell. Moreover, the term “vector” may also refer to at least onepolynucleotide formulated with a preparation of liposomes or lipidnanoparticles which is capable of transfecting a cell with the at leastone polynucleotide as described, e.g. by Geall et al., 2012, PNAS,109:14604-14609.

At least one polynucleotide comprised by the vector consists of orcomprises at least one nucleic acid construct encoding at least oneimmunogenic protein. In addition to the polynucleotide consisting of orcomprising the nucleic acid construct of the present inventionadditional polynucleotides and/or polypeptides may be introduced intothe cell. The addition of additional polynucleotides and/or polypeptidesis also contemplated if said additional polynucleotides and/orpolypeptides are required to introduce the nucleic acid construct of thepresent invention into the cell or if the introduction of additionalpolynucleotides and/or polypeptides increases the expression of theimmunogenic polypeptide encoded by the nucleic acid construct of thepresent invention.

In the context of the present invention it is envisioned that theantigenic protein or the antigenic fragment thereof encoded by theintroduced nucleic acid construct are expressed within the cell uponintroduction of the vector or vectors. Examples of suitable vectorsinclude but are not limited to plasmids, cosmids, phages, viral vectors,lipid nanoparticles or artificial chromosomes.

In an embodiment of the present invention the viral vector is selectedfrom the group consisting of adenovirus vectors, adeno-associated virus(AAV) vectors (e.g., AAV type 5 and type 2), alphavirus vectors (e.g.,Venezuelan equine encephalitis virus (VEE), sindbis virus (SIN), semlikiforest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g.vectors derived from cytomegaloviruses, like rhesus cytomegalovirus(RhCMV), arena virus vectors (e.g. lymphocytic choriomeningitis virus(LCMV) vectors), measles virus vectors, poxvirus vectors, paramixovirusvector, baculovirus vectot vesicular stomatitis virus vectors,retrovirus, lentivirus, viral like particles, and bacterial spores.

In a further embodiment, vectors are adenoviral vectors, in particularadenoviral vectors derived from human or non-human great apes. Exemplarygreat apes from which the adenoviruses are derived are Chimpanzee (Pan),Gorilla (Gorilla) and orangutans (Pongo), for example Bonobo (Panpaniscus) and common Chimpanzee (Pan troglodytes). Typically, naturallyoccurring non-human great ape adenoviruses are isolated from stoolsamples of the respective great ape. Specifically, vectors arenon-replicating adenoviral vectors based on hAd5, hAd11, hAd26, hAd35,hAd49, ChAd3, ChAd4, ChAd5, ChAd6, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11,ChAd16, ChAd17, ChAd19, ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31,ChAd37, ChAd38, ChAd44, ChAd55, ChAd63, ChAd 73, ChAd82, ChAd83,ChAd146, ChAd147, PanAd1, PanAd2, and PanAd3 vectors orreplication-competent Ad4 and Ad7 vectors. The human adenoviruses hAd4,hAd5, hAd7, hAd11, hAd26, hAd35 and hAd49 are well known in the art.Vectors based on naturally occurring ChAd3, ChAd4, ChAd5, ChAd6, ChAd7,ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19, ChAd20, ChAd22,ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44, ChAd63 andChAd82 are described in detail in WO 2005/071093, which published as US20110217332 and is incorporated by reference as to the adenoviralvectors described therein. Vectors based on naturally occurring PanAd1,PanAd2, PanAd3, ChAd55, ChAd73, ChAd83, ChAd146, and ChAd147 aredescribed in detail in WO 2010/086189, which published as US 20120027788and is incorporated by reference as to the adenoviral vectors describedtherein.

In another embodiment, the second antigenic protein or antigenicfragment thereof is immunologically identical to the antigenic proteinor antigenic fragment thereof encoded by the nucleic acid constructcomprised by the poxviral vector.

In another embodiment of the present invention, the vector is present asnaked DNA. The term “naked DNA” refers to any nucleic acid molecule, DNAor RNA, which does not encode proteins of a viral vector but encodes atleast one antigenic protein or fragment thereof. It is contemplated thatnaked DNA is not associated with any polypeptides, in particular notwith polypeptides of viral origin. For example, naked DNA is present asplasmid, cosmid or as an artificial chromosome. In a further embodiment,the naked DNA encodes a polypeptide which is immunologically identicalto the antigenic protein or antigenic fragment thereof encoded by thenucleic acid construct comprised by the poxviral vector.

The term “viral like particle” (VLP) refers to assemblies comprisingviral proteins but no nucleic acid. VLPs can be produced by expressingviral surface proteins in suitable producer cell-lines. The lack ofnucleic acid, and thus genetic information, renders VLP non-infectious,thus creating a safe vaccine. For example, the VLP comprises apolypeptide which is immunologically identical to the antigenic proteinor antigenic fragment thereof encoded by the nucleic acid constructcomprised by the poxviral vector.

In a certain embodiment of the present invention the above-describedvaccine combination is used in a prime-boost vaccination regimen. In afirst embodiment of this prime-boost vaccination regimen the poxviralvector is used for priming the immune response and the viral vector orantigenic protein or antigenic fragment thereof is used for boosting theimmune response. In another embodiment of the prime-boost vaccinationregimen, the viral vector or antigenic protein or antigenic fragmentthereof is, used for priming the immune response and the poxviral vectoris used for boosting the immune response.

In an embodiment of the present invention,

-   -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one oral        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one topical        administration;    -   the immune response is primed by intranasal administration and        the immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one oral        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one topical        administration;    -   the immune response is primed by intramuscular administration        and the immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one oral        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one topical        administration;    -   the immune response is primed by subcutaneous administration and        the immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one oral        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one topical        administration;    -   the immune response is primed by intradermal administration and        the immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one oral        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one topical        administration;    -   the immune response is primed by intragastric administration and        the immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one oral administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one topical        administration;    -   the immune response is primed by oral administration and the        immune response is boosted by at least one intranasal        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one intramuscular        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one subcutaneous        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one intradermal        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one intragastric        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one oral administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one topical        administration;    -   the immune response is primed by topical administration and the        immune response is boosted by at least one intranasal        administration.

In one embodiment, the immune response is primed by intranasaladministration and the immune response is boosted by at least oneintramuscular administration.

In yet another embodiment, the immune response is primed by intranasaladministration and the immune response is boosted by at least oneintranasal administration.

In yet another embodiment, the immune response is primed byintramuscular administration and the immune response is boosted by atleast one intramuscular administration.

In a further aspect, the present invention relates to a vaccinecomposition comprising a poxviral vector for priming an immune responseas defined above or a vaccine combination comprising a poxviral vectorand an agent selected from the group consisting of (i) a vectorcomprising a nucleic acid sequence encoding at least a second antigenicprotein or antigenic fragment thereof, (ii) a second antigenic proteinor antigenic fragment thereof and (iii) viral like particles.

The term “composition” refers to the combination comprising an antigenicprotein or fragment thereof or a viral-like particle or vectorcomprising a nucleic acid construct and at least one further compoundselected from the group consisting of pharmaceutically acceptablecarriers, pharmaceutical excipients and adjuvants.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier”, as used herein, refers to a pharmacologicallyinactive substance such as but not limited to a diluent, excipient, orvehicle with which the therapeutically active ingredient isadministered. Such pharmaceutical carriers can be liquid or solid.Liquid carrier include but are not limited to sterile liquids, such assaline solutions in water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. A saline solution is onepreferred carrier when the pharmaceutical composition is administeredintravenously or intranasally by a nebulizer.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like.

Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

The term “adjuvant” refers to agents that augment, stimulate, activate,potentiate, or modulate the immune response to the active ingredient ofthe composition at either the cellular or humoral level, e g immunologicadjuvants stimulate the response of the immune system to the actualantigen, but have no immunological effect themselves. Examples of suchadjuvants include but are not limited to inorganic adjuvants (e.g.inorganic metal salts such as aluminium phosphate or aluminiumhydroxide), organic adjuvants (e.g. saponins or squalene), oil-basedadjuvants (e.g. Freund's complete adjuvant and Freund's incompleteadjuvant), cytokines (e.g. IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS, andINF-γ) particulate adjuvants (e g immuno-stimulatory complexes (ISCOMS),liposomes, or biodegradable microspheres), virosomes, bacterialadjuvants (e.g. monophosphoryl lipid A, or muramyl peptides), syntheticadjuvants (e.g. non-ionic block copolymers, muramyl peptide analogues,or synthetic lipid A), or synthetic polynucleotides adjuvants (e.gpolyarginine or polylysine).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: MVA encoding Invariant chain (li)—NS antigen induces stronger Tcell response in mice. Two groups of mice were immunized with 2×10^5plaque forming units (pfu) of MVA comprising NS-antigen (left panel) andwith MVA comprising NS linked to invariant chain (right panel). Total Tcell response to NS antigen was measured by IFNγ ELISpot and numbers ony axis represent spot forming cells (SFC)/million splenocytes.

FIG. 2: MVA encoding Invariant chain (li)—NS antigen induces stronger Tcell response than the corresponding Adeno in mice. Two groups of micewere immunized with MVA encoding NS (MVA wt) or NS linked to humaninvariant chain (MVAli). Two additional groups of mice were immunizedwith a comparable dose of ChAd3 encoding NS (ChAd3 wt) or NS linked tohuman invariant chain (ChAd3li). Total T cell response to NS antigen wasmeasured by IFNγ ELISpot and numbers on y axis represent spot formingcells (SFC)/million splenocytes.

FIG. 3: Boosting with MVA comprising NS linked to invariant chainaugments the generation of HCV-NS specific T cells in macaques.

Two groups of 4 macaques were primed with ChAd3IiNS and 50 weeks laterboosted with MVA-NS (grey bars) or with MVA-IiNS (black bars). Panel Ashows the response by IFNγ ELlspot one week (peak boost) or 3 monthspost boost (memory). Numbers on y axis represent spot forming cells(SFC)/million PBMC. Panel B shows higher CD8 frequency by IFNγ ICS oneweek post boost with MVAIiNS (black bars). Numbers on y axis represent %of antigen-specific CD8 T cells producing IFNγ.

FIG. 4: Schematic diagram showing the four isoforms of human invariantchain (p33, p35, p41, p43, isoform c) and the two isoforms of murineinvariant chain (p31, p41). In human p35 and p43 an additional 16residues are present at the N-terminus due to alternative initiation oftranslation. In human p41 and p43 isoforms and the murine p41 isoform anadditional domain is present due to alternative splicing. The human cisoform lacks two exons relative to human p33 and p35 (three exonsrelative to human p41 and p43) leading to a frame-shift.

EXAMPLES Example 1 Priming with MVA Comprising NS Linked to InvariantChain (MVA-hli NS) Augments the Generation of HCV-NS Specific T Cells inMice

Two groups of Balb/c mice were immunized intramuscularly with 2×10^5 pfu(plaque forming units) of MVA encoding NS or with the same dose of MVAcomprising NS linked to human invariant chain. The NS region encompassesabout two thirds of the HCV genome and encodes for five differentproteins (NS3, NS4A, NS4B, NS5A and NS5B) that result from theproteolytic cleavage of the HCV polyprotein by the encoded NS3 protease.Ten days after immunization, splenocytes were collected and HCV-NSspecific T cell response was evaluated by IFNγ ELlspot using pools ofpeptides spanning NS. The response was evaluated by summing upreactivities against the six individual peptide pools and subtractingbackground (spots counted in control wells with no peptide). The levelof specific T cells targeting NS was higher in mice primed with theli-based MVA vaccine (FIG. 1).

Example 2 Priming with MVA Comprising NS Linked to Invariant Chain(MVA-Hli NS) Induces Stronger T Cell Response in Mice than theCorresponding Adenoviral Vector

Two groups of Balb/c mice were immunized intramuscularly with 2×10^5 pfuof MVA encoding NS or with the same dose of MVA comprising NS linked tohuman invariant chain. Two additional groups of mice were immunized with2×10^5 iu (infective units) of ChAd3 encoding NS or with the same doseof ChAd3 comprising NS linked to human invariant chain. Peak immuneresponse was evaluated on splenocytes collected 10 and 21 days afterimmunization with MVA and ChAd3 vectored vaccines, respectively. T cellresponse was evaluated by IFNγ ELlspot using pools of peptides spanningNS. The results (FIG. 2) show that the li-based MVA vaccine induceshigher response than the corresponding li-based ChAd3 vaccine.

Example 3 Boosting with MVA Comprising NS Linked to Invariant Chain(MVA-Hli NS) Augments the Generation of HCV-NS Specific T Cells inMacaques

Two groups of 4 macaques were primed with ChAd3IiNS and 50 weeks laterboosted with MVA-NS (grey bars) or with MVA-IiNS (black bars). Theinjected dose was 1×10¹⁰ vp for adenoviral vectors, and 2×10⁸ pfu forMVA vectors Immune response was evaluated on PBMC collected 1 week (peakresponse) and 3 months (memory response) after priming by IFNγ ELlspotand IFNγ Intracellular staining (ICS) using pools of peptides spanningNS. As shown in FIG. 3, higher ELISpot response was induced in the groupreceiving MVAIiNS at both time points (black bars). Panel A shows theresponse by IFNγ ELlspot one week or 3 months post boost. Panel B showshigher frequency of CD8 T cells producing IFNγ by ICS one week postboost with MVAIiNS (black bars).

Materials and methods

Adenoviral and MVA vectors

The ChAd3 vector expressing the entire HCV NS3-5B (NS) region fromgenotype 1b, strain bk, has been described previously (Colloca et al.Sci Transl Med 4(115), 115ra112, 2012). MVA vector expressing the samecassette was derived and prepared as described previously (Cottingham,M. G. et al PLoS ONE 3, e1638, 2008; Di Lullo, G. et al. Virol. Methods156, 37-43, 2009). The human Ii (p35, NCBI Reference Sequence:NM_004355) insert was synthetized by GeneArt (Life Technologies,Paisley, UK) and then cloned at the N-terminus of the NS transgene underHCMV and BGHpA control.

Animals and vaccinations

All experimental procedures were performed in accordance with nationaland international laws and policies (EEC Council Directive 86/609;Italian Legislative Decree 116/92). The ethical committee of the ItalianMinistry of Health approved this research Animal handling procedureswere performed under anesthesia and all efforts were made to minimizesuffering and reduce animal numbers. Female 6-week-old Balb/c or C57B1/6mice were purchased from Charles River (Como, Italy), and experimentalgroups of 5 mice each were set. ChAd3 and MVA vectors were administeredintramuscularly in the quadriceps by delivering a volume of 50 μl persite (100 μl final volume).

Naïve, female, 11 to 19 years old (weight range 3.2 to 6.5 Kg)Cynomolgus macaques (macaca fascicularis) from a purpose bred colonyhoused at the Institute of Cell Biology and Neurobiology (NationalResearch Council of Italy, Rome), were assigned to experimental groupsof four animals each. All immunizations were delivered by intramuscularroute in the deltoid muscle injecting 0.5 ml of virus diluted instabilizing buffer. The injected dose was 1×10¹⁰ vp for adenoviralvectors, and 2×10⁸ pfu for MVA vectors. During handling, the animalswere anesthetized by i.m. injection of 10 mg/kg ketamine hydrochloride.

Peptides

A set of 494 peptides, 15 amino acids in length, overlapping by 11 aminoacids and spanning the open reading frame from NS3-NS5B (1985 a.a.) ofHCV genotype 1b strain BK were obtained from BEI Resources (Manassas,Va.).

Ex vivo IFNγ ELISpot with mouse and macaque samples

MSIP 54510 plates (Millipore) were coated with 10 μg/ml of anti-mouse oranti-monkey IFNγ antibody (both from U-CyTech Utrecht, The Netherlands)overnight at 4° C. After washing and blocking, mouse splenocytes ormacaque peripheral blood mononuclear cells (PBMC) were plated induplicate at two different densities (2×10⁵ and 4×10⁵ cells/well) andstimulated overnight with overlapping 15mer peptide pools at a finalconcentration of 4 μg/ml each single peptide. The peptide diluent DMSO(Sigma-Aldrich, Milan, Italy) and ConA (Sigma-Aldrich, Milan, Italy)were used respectively as negative and positive controls. Plates weredeveloped by subsequent incubations with biotinylated anti-mouse oranti-monkey IFNγ antibody (both from U-CyTech Utrecht, The Netherlands),Streptavidin-Alkaline Phosphatase conjugated (BD Biosciences, NJ) andfinally with BCIP/NBT 1-Step solution (Thermo Fisher Scientific,Rockford, Ill.). Plates were acquired and analyzed by an A.EL.VISautomated plate reader. The ELISpot response was considered positivewhen all of the following conditions were met: IFNγ production presentin Con-A stimulated wells; at least 50 specific spots/millionsplenocytes or PBMC to at least one peptide pool; the number of spotsseen in positive wells was three times the number detected in the mockcontrol wells (DMSO); and that responses decreased with cell dilutions.ELISpot data were expressed as IFNγ spot forming cells (SFC) per millionsplenocytes or PBMC.

Intracellular Cytokine Staining (ICS) and FACS analysis with macaquesamples

Briefly, 2×10⁶ monkey PBMCs were stimulated at 37° C. in 5% CO₂ for15-20 hours using peptide pools as antigen at 2 μg/ml each peptide finalconcentration in presence of anti-human CD28/CD49d costimulatoryantibodies (BD Biosciences, NJ) and Brefeldin A (Sigma-Aldrich, Milan,Italy). DMSO (Sigma-Aldrich, Milan, Italy) was used as negative control,and Staphylococcal enterotoxin B (SEB, Sigma-Aldrich, Milan, Italy) wasused as positive control. After overnight stimulation, PBMCs wherestained with the following surface antibodies: APC anti-monkey CD3,clone SP34-2; PerCp-Cy5.5 anti-monkey CD4, clone L200; PE anti-humanCD8, clone RPA-T8 (all from BD Biosciences, NJ). Intracellular stainingwas performed after treatment with Cytofix/Cytoperm and in the presenceof PermWash (BD Biosciences, NJ) using FITC anti-human IFNγ, clone MD-1(U-CyTech Utrecht, The Netherlands). Stained cells were acquired on aFACS Canto flow cytometer, and analyzed using DIVA software (BDBiosciences, NJ). At least 30,000 CD8+, CD3+ gated events were acquiredfor each sample.

The invention claimed is:
 1. A Modified Vaccinia Ankara virus comprisinga nucleic acid construct comprising: (i) a nucleic acid sequenceencoding at least one invariant chain operatively linked to (ii) anucleic acid encoding at least one antigenic protein or antigenicfragment thereof; wherein the C-terminus of the encoded invariant chainis directly or indirectly linked to the N-terminus of the encodedantigenic protein or antigenic fragment thereof.
 2. The ModifiedVaccinia Ankara virus according to claim 1, wherein the at least oneencoded invariant chain is of mammalian origin.
 3. The Modified VacciniaAnkara virus according to claim 1, wherein the encoded at least oneinvariant chain is characterized by at least one of the followingfeatures: (i) the endogenous KEY-region is deleted or substituted by adifferent sequence; (ii) the methionine in positions 107 and 115 (humaninvariant chain) or in positions 90 and 98 (murine invariant chain) orthe positions corresponding thereto in another invariant chain issubstituted by another amino acid; (iii) at least one sorting peptide isadded to, removed from or replaces the endogenous sorting peptide of theinvariant chain, and/or (iv) at least one Class II-associated li chainpeptide (CLIP) region is added to, removed from or replaces theendogenous CLIP region of the at least one invariant chain.
 4. TheModified Vaccinia Ankara virus according to claim 1, wherein the encodedat least one invariant chain is a fragment of SEQ ID NO: 1 of at least40 consecutive amino acids or has at least 85% sequence identity to thesame fragment of SEQ ID NO:
 1. 5. The Modified Vaccinia Ankara virusaccording to claim 1, wherein the at least one antigenic protein is aprotein of a pathogenic organism.
 6. The Modified Vaccinia Ankara virusaccording to claim 5, wherein the pathogenic organism is a virus, abacterium, a protist or a multicellular parasite.
 7. The ModifiedVaccinia Ankara virus according to claim 1, wherein the priming of animmune response is part of a homologous prime-boost vaccination regimen.8. The Modified Vaccinia Ankara virus according to claim 1, wherein thepriming of an immune response is part of a heterologous prime-boostvaccination regimen.
 9. The Modified Vaccinia Ankara virus according toclaim 1, wherein the Midified Vaccinia Ankara virus is administered viaintranasal, intramuscular, subcutaneous, intradermal, intragastric, oralor topical routes.
 10. A method for stimulating an immune responsecomprising administering to a subject a composition comprising theModified Vaccinia Ankara virus of claim
 1. 11. A Modified VacciniaAnkara virus comprising a nucleic acid construct for priming or boostingan immune response, the nucleic acid construct comprising: (a) a nucleicacid sequence encoding at least one invariant chain operatively linkedto (b) a nucleic acid encoding at least one antigenic protein orantigenic fragment thereof wherein the C-terminus of the encodedinvariant chain is directly or indirectly linked to the N-terminus ofthe encoded antigenic protein or antigenic fragment thereof, and whereinafter administration to a subject the CD8+ T cell response of thesubject against the antigenic protein or antigenic fragment thereof isincreased as compared to the same regimen without invariant chain; andwherein the virus increases the antigen specific CD8+ T cell response ina subject.
 12. A Modified Vaccinia Ankara virus of claim 1, wherein theinvariant chain is selected from p35 or a variant or fragment thereof.